作業は、トランスレーショナル医学と治療のための研究所からのフェローシップ、およびペンシルベニア大学で医学と工学（T32HL007954）研究所から訓練助成金とマレーネL.コーエンとでジェロームH. Fleisch学者グラントによって資金を供給されたコネチカット大学保健センター（LHS）とNIH NS - 029331（FAW）。

1。機器の準備<ol><li>細菌汚染を最小限に抑えるために75％エタノールで定位フレームとポンプを拭う。 </li><li>ハミルトンシリンジとフューズドシリカニードルを滅菌する。 注：化学物質の滅菌が使用されている場合は、使用前に滅菌水で数回洗浄してください。 </li><li> 75％エタノールでパラフィンワックスペーパーの表面を拭き、乾燥させてください。 </li></ol> 2。注射のためのマウスの準備注意：それらが新しい環境に順応し、ストレスを軽減できるように、少なくとも7日間手術前に、動物施設に供給されるマウスを持っている。 <ol><li>術前のベースラインのためのマウスの重量を量る。 </li><li>テールピンチに応答するまで、イソフルラン30％酸素、70％亜酸化窒素、および4％で麻酔を誘発する</li><li>術後鎮痛のためにブプレノルフィン0.1mg/kg腹腔内にマウスを注入する</li><li>頭皮を剃る</li><li>無菌石油ゼリーでコーティングの目</li><li> betadine × 3ワイプと頭皮を準備して、頭皮が乾燥することができます</li><li>滅菌外科用メスを使用して頭皮の1cm正中矢状切開を加えます 注：寛大な切開が頭蓋骨のランドマークの完全な露出をできるようになります。 </li><li>ベースから1cmとbetadineで準備× 3ワイプを開始する尾の腹側表面の1cmを剃る</li><li>定位フレームの上にマウスを置きます 注：これは、マウスが優れたブレグマの曝露とブレグマの右に3 mm以上で、フレームのベースと頭蓋骨の平行の表面でフレームに固定されるようにすることが重要です。 </li></ol> 3。脳内出血の手術注：全体の手術中にマウスが連続して37℃に維持イソフルラン30％酸素、70％亜酸化窒素、および1-3％、± 0.5℃のサーミスタ制御の加熱パッドを使用し、直腸温度計によって監視で麻酔です。 <ol><li>フレームに1 ccシリンジに滅菌27グラムの針を取り付けます。 </li><li>針が正確にブレグマ以上になるまで、定位固定アームを調整します。 </li><li>その針はブレグマの外側と頭蓋骨の表面に低い2.5ミリメートルになるようにアームを調整します。 </li><li>手動で完全に頭蓋骨に穴をしないフレームテイクケアの穏やかな下方への移動を印加しながら頭蓋骨の表面にバリ穴を作るために注射器を回転させる。 </li><li>注射器/注射針を使用して手動で針と完全なバリ穴を削除します。 注：あなたが頭蓋骨と誤って脳実質に針を押すのを最小限に抑えるリスクの内部表を穴あきがある場合、手作業でバリ穴を完了するには、即時認識することができます。 </li><li>滅菌外科用ブレードを用いて尾の腹面に横切開を行い、動脈血の2-3大滴はパラフィンワックスペーパーの上に落ちるように。その後すぐに滅菌ガーゼを用いて、圧力で出血を止める。 </li><li>ポンプでハミルトンシリンジと場所のシリンジに17μLの血液を撤回。 </li><li>ポイント5に定位固定アームを調整して°の垂直軸に対して内側に相対的。 </li><li>針のその先端が頭蓋骨のバリ穴と、下部針3.5ミリメートルを超えているので、慎重に定位固定アームを調整する。 </li><li> 2分後、針0.5mmの撤退を待つ（ように先端が深さ3mmである） </li><li>脳は、針の周りに再展開し、注入時の針挿入のトラックを還流血液の危険性を最小限にするように5分間待ちます。 </li><li> 7.5μLの血液1μL/分を注入する。 </li><li>初期の血液凝固を可能にすると組織の変化のために頭蓋内圧の上昇を最小限に抑えるために発生する5分待ってください。 </li><li> 1μL/分で、残りの7.5μLを注入</li><li>針は、血液凝固を可能にするために25分のための場所の中に残さないように 注：血液凝固を待つに失敗すると、針を引き出す時に血液が針の挿入部位を還流につながる</li><li>ゆっくりと針を撤回し、直ちに凝固から針での残留血液を予防し、針の再利用性を確保するためにお湯で洗い流してください。 </li><li>フレームからマウスを削除し、獣医外科的接着剤で尾と頭皮切開を閉じてください。 </li><li>麻酔の電源を切ります。 </li><li>継続的に食物を湿らせて自由にアクセスできるように暖めている間に目覚めさせるマウスすることができます。 </li><li>完全に目覚め同腹でケージにマウスを返します。食料へのアクセスに動物を助けるためにケージの底部に水分のある食品ペレットを置きます。 </li></ol> 4。代表的な結果： <img src="/files/ftp_upload/2618/2618fig1.jpg" alt="図1"/> 図1 15分ICHの手術後のマウス脳の冠状断面。直ちに脳を犠牲にした後は、針の挿入部位での冠状断面の総点検に基づいて、ICHの成功のために検査した。脳梁過去針のトラックまで、または心室に、脳の基底部にダウン追跡出血が失敗したとみなされ、そのマウスは、すべての分析から除外した。全体的なICH succeSS率が0％の死亡率で50匹に75から85パーセントでした。 <img src="/files/ftp_upload/2618/2618fig2.jpg" alt="図2"/> 図2。シリンダーテストでは、右大脳基底核のICHの後に左片麻痺を示しています。 ICHの手術後の（A）サンプルマウスの背面。左大脳基底核のICH後のシリンダーの壁にのみ右前肢の配置に注意してください。 （B）シャム（N = 4）と比較してICH手術（n = 5）は後のマウスのコホートから円筒試験1の結果のグラフを。偽のマウスは、血液の注射（針を脳に挿入された）を除いてすべての手順を持っていた。各マウスは、12 cmの直径の透明ガラス製のシリンダーに入れ、20後部のために観察された。シリンダーの壁に前肢の初期配置はリアごとに採点した。マウスが地面に戻り、次の後部がゴールするまで、後続の動きは（例えば横方向探査など）採点されていなかった。側性指数は、（シリンダーの側面に＃右前肢の配置 - ＃左前肢のプレースメント）として算出した（＃右+＃左+＃両方）/、0は右前肢が使用されたのみ示されているも前肢優先と1を示していない場所。

<ol>
	<li>Hua, Y., Schallert, T., Keep, R. F., Wu, J., Hoff, J. T., Xi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+Tests+After+Intracerebral+Hemorrhage+in+the+Rat.">Behavioral Tests After Intracerebral Hemorrhage in the Rat.</a> Stroke. 33, 2478-2484 (2002).</li><li>James, M., Warner, D., Laskowitz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+Models+of+Intracerebral+Hemorrhage:+A+Translational+Perspective.">Preclinical Models of Intracerebral Hemorrhage: A Translational Perspective.</a> Neurocritical Care. 9, 139-152 (2008).</li><li>Foerch, C., Arai, K., Jin, G., Park, K. -. P., Pallast, S., van Leyen, K., Lo, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Model+of+Warfarin-Associated+Intracerebral+Hemorrhage.">Experimental Model of Warfarin-Associated Intracerebral Hemorrhage.</a> Stroke. 39, 3397-3404 (2008).</li><li>Nakamura, T., Xi, G., Hua, Y., Schallert, T., Hoff, J. T., Keep, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+hemorrhage+in+mice:+model+characterization+and+application+for+genetically+modified+mice.">Intracerebral hemorrhage in mice: model characterization and application for genetically modified mice.</a> Journal of Cerebral Blood Flow and Metabolism. 24, 487-494 (2004).</li><li>Rynkowski, M. A., Kim, G. H., Komotar, R. J., Otten, M. L., Ducruet, A. F., Zacharia, B. E., Kellner, C. P., Hahn, D. K., Merkow, M. B., Garrett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+intracerebral+hemorrhage+using+autologous+blood+infusion.">A mouse model of intracerebral hemorrhage using autologous blood infusion.</a> Nat. Protocols. 3, 122-128 (2008).</li><li>Wang, J., Fields, J., Dor&#233;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+an+improved+preclinical+mouse+model+of+intracerebral+hemorrhage+using+double+infusion+of+autologous+whole+blood.">The development of an improved preclinical mouse model of intracerebral hemorrhage using double infusion of autologous whole blood.</a> Brain Research. 1222, 214-221 (2008).</li><li>Zhao, X., Sun, G., Zhang, J., Strong, R., Song, W., Gonzales, N., Grotta, J. C., Aronowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoma+resolution+as+a+target+for+intracerebral+hemorrhage+treatment:+Role+for+peroxisome+proliferator-activated+receptor+&#947;+in+microglia/macrophages.">Hematoma resolution as a target for intracerebral hemorrhage treatment: Role for peroxisome proliferator-activated receptor &#947; in microglia/macrophages.</a> Annals of Neurology. 61, 352-362 (2007).</li></ol>

利害の衝突は宣言されません。

自発的な基底核出血の再現可能なモデルで自家尾動脈の血液の結果を使用してマウスの脳内出血のこの外科的モデル。マウスにおけるICHのモデルは、病態生理を調査するためにトランスジェニック動物の可用性の利点を提供しているが、その小さなサイズが大きい動物よりも脳神経外科手術はより技術的に困難になります。 コラゲナーゼのモデルと自家血注入モデルは?...

マウスの脳神経外科のための定位フレーム（Stoelting、51925） Microinfusionポンプとプロセッサ（UMP - 3とMicro4、世界の精密機器、サラソタ、フロリダ州） マウスウォーマー（Stoelting、50300） 吸入マウスの麻酔（ブレーントリーサイエンティフィック、EZ - AF9000） コー​​ティングされたプランジャーとのない針で25μL気密ホウケイ酸ハミルトンシリンジ （ハミルトン社、リノ、ネバダ州、1702RNのシリンジ：765401、フェルール：30949、スペーサー：30946） フューズドシリカニードルは2cmの長さ（ハミルトン、17739）にカット *前に、各手術に滅菌場合ハミルトンシリンジとフューズドシリカニードルが複数の手術のために再利用化されることがあります。これらの材料は、血液凝固を避けるために不可欠です。 無菌手術用手袋手術衣、ボンネットとマスク Betadine 75％エタノール滅菌27グラム針（シングルユース） 滅菌1 ccシリンジ（シングルユース） 滅菌外科用ブレード Cidex 滅菌水ブプレノルフィンとイソフルラン滅菌ガーゼパラフィンワックスペーパーの正方形獣医外科接着剤（Vetbond、3M、セントポール、ミネソタ州）

autologous blood injection to model spontaneous intracerebral hemorrhage in mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

Watch this Scientific Journal Video about Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice at JoVE.com

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

このプロトコルで説明されているマウスの脳内出血の自家血注入モデルでは、針のトラックまでの血液逆流、ポンプシステムにおける無抗凝固剤のリスクを最小限に抑えるために二重注入の技術を使用し、システム内のすべてのデッドスペースと拡張可能なチューブを排除。

マウスにおける自発脳内出血をモデル化する自家血注入

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

autologous-blood-injection-to-model-spontaneous-intracerebral

Research

JoVE Journal

Neuroscience

14.7K ビュー.  University of Connecticut Health Center. このプロトコルで説明されているマウスの脳内出血の自家血注入モデルでは、針のトラックまでの血液逆流、ポンプシステムにおける無抗凝固剤のリスクを最小限に抑えるために二重注入の技術を使用し、システム内のすべてのデッドスペースと拡張可能なチューブを排除。

ビデオ: Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson&#8217;s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies&#8217; ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents.

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, causing locomotion defects. Rotenone models Parkinson&#8217;s disease in Drosophila. This paper outlines two assays that characterize both spontaneous and startle-induced locomotion deficiencies caused by rotenone.

のロテノン誘発性のパーキンソン病モデルにおける自発と驚愕誘発性歩行を特徴づけるための方法<em&gt;ショウジョウバエ</em

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

神経科学

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

急性アルツハイマーなどの認知障害を誘発するために正常マウスにおけるアミロイドβペプチドの脳室内注射

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

脳内注入戦略を使用してジカ ウイルス性神経疾患のマウスモデルの確立: 胎生期、新生児、およびアダルト

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

炎症性サイトカインの脳内注射による広範囲にわたる大脳皮質脱髄のラットモデル

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

くも膜下出血のマウスモデルにおける脳血管攣縮の定量化法の体積

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Minimally Invasive Endoscopic Intracerebral Hemorrhage Evacuation

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and poor functional outcomes, largely because there are no evidence-based treatment options for this devastating disease process. In the past decade, a number of minimally invasive surgeries have emerged to address this issue, one of which is endoscopic evacuation. Stereotactic ICH Underwater Blood Aspiration (SCUBA) is a novel endoscopic evacuation technique performed in a fluid-filled cavity using an aspiration system to provide an additional degree of freedom during the procedure. The SCUBA procedure utilizes a suction device, endoscope, and sheath and is divided into two phases. The first phase involves maximal aspiration and minimal irrigation to decrease clot burden. The second phase involves increasing irrigation for visibility, decreasing aspiration strength for targeted aspiration without disturbing the cavity wall, and cauterizing any bleeding vessels. Using the endoscope and aspiration wand, this technique aims to maximize hematoma evacuation while minimizing collateral damage to the surrounding brain. Advantages of the SCUBA technique include the use of a low-profile endoscopic sheath minimizing brain disruption and improved visualization with a fluid-filled cavity rather than an air-filled one.

This paper details the surgical protocol for minimally invasive endoscopic intracerebral hemorrhage evacuation using the SCUBA technique.

低侵襲内視鏡性脳内出血

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and poor functional outcomes, largely because there are no evidence-based ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

成体マウスの遺伝子組換えアデノ随伴ウイルスの直接髄腔内注入

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

ウイルスベクターの脳内注射によるインビボグリア細胞のインターニューロンへの直接リプログラミング

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

くも膜下出血のモデルとしての水槽マグナへの血液の二重直接注射

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 &#181;L of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.

We present a protocol to establish a massive pontine hemorrhage model in a rat via dual injection of autologous blood.

マウスにおける自発脳内出血をモデル化する自家血注入

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この動画の章

のロテノン誘発性のパーキンソン病モデルにおける自発と驚愕誘発性歩行を特徴づけるための方法

急性アルツハイマーなどの認知障害を誘発するために正常マウスにおけるアミロイドβペプチドの脳室内注射

脳内注入戦略を使用してジカウイルス性神経疾患のマウスモデルの確立: 胎生期、新生児、およびアダルト

炎症性サイトカインの脳内注射による広範囲にわたる大脳皮質脱髄のラットモデル

くも膜下出血のマウスモデルにおける脳血管攣縮の定量化法の体積

低侵襲内視鏡性脳内出血

成体マウスの遺伝子組換えアデノ随伴ウイルスの直接髄腔内注入

ウイルスベクターの脳内注射によるインビボグリア細胞のインターニューロンへの直接リプログラミング

くも膜下出血のモデルとしての水槽マグナへの血液の二重直接注射

自家血液の二重注射による巨大なポンチン出血

マウスにおける自発脳内出血をモデル化する自家血注入

要約

さらに動画を探す

この動画の章

のロテノン誘発性のパーキンソン病モデルにおける自発と驚愕誘発性歩行を特徴づけるための方法

急性アルツハイマーなどの認知障害を誘発するために正常マウスにおけるアミロイドβペプチドの脳室内注射

脳内注入戦略を使用してジカ ウイルス性神経疾患のマウスモデルの確立: 胎生期、新生児、およびアダルト

炎症性サイトカインの脳内注射による広範囲にわたる大脳皮質脱髄のラットモデル

くも膜下出血のマウスモデルにおける脳血管攣縮の定量化法の体積

低侵襲内視鏡性脳内出血

成体マウスの遺伝子組換えアデノ随伴ウイルスの直接髄腔内注入

ウイルスベクターの脳内注射によるインビボグリア細胞のインターニューロンへの直接リプログラミング

くも膜下出血のモデルとしての水槽マグナへの血液の二重直接注射

自家血液の二重注射による巨大なポンチン出血

脳内注入戦略を使用してジカウイルス性神経疾患のマウスモデルの確立: 胎生期、新生児、およびアダルト